Gene Cloning

Cloning - a definition
 From the Greek - klon, a twig
 An aggregate of the asexually produced progeny
of an individual;a group of replicas of all or part
of a macromolecule (such as DNA or an antibody)
 An individual grown from a single somatic cell of
its parent & genetically identical to it
 Clone: a collection of molecules or cells, all
identical to an original molecule or cell
 DNA CLONING

A method for identifying and purifying a

particular DNA fragment (clone) of interest
from a complex mixture of DNA fragments,
and then producing large numbers of the
fragment (clone) of interest.
Gene cloning
 When DNA is
extracted from an
organism, all its genes
are obtained
 In gene (DNA)
cloning a particular
gene is copied
(cloned)
 Why Clone DNA?
 A particular gene can be isolated and its nucleotide
sequence determined
 Control sequences of DNA can be identified &
analyzed
 Protein/enzyme/RNA function can be investigated
 Mutations can be identified, e.g. gene defects
related to specific diseases
 Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect resistance,
etc.
Sources of DNA for Cloning

 1) Chromosomal DNA

 2) RNA converted to cDNA

 4) PCR-amplified DNA
PCR-amplified DNA
Cloning Tools
 Restriction endonucleases
 Ligase
 Vectors
 Host
 Methods for introducing DNA into a host cell
Cutting DNA

 Restriction endonucleases
(restriction enzymes)
• sticky ends
• blunt ends

 Nomenclature
• EcoRI
• E = genus (Escherichia)
• co = species (coli)
• R = strain
• I = # of enzyme
Blunt & Sticky ends
Pasting DNA

 Complementary ends
(sticky ends) H-bond

 Ligase forms
phosphodiester bond
to seal strands
together.
Cloning vectors

allowing the exogenous DNA to be

inserted, stored, and manipulated
mainly at DNA level.
1 Plasmid vectors
2 Bacteriophage vectors
3 Cosmids
Plasmid vectors

Plasmid vectors are double-stranded, circular, self-

replicating, extra-chromosomal DNA molecules.
 Advantages:
• Small, easy to handle
• Straightforward selection strategies
• Useful for cloning small DNA fragments
(< 10kbp)
 Disadvantages:
• Less useful for cloning large DNA fragments
(> 10kbp)
 A plasmid vector for cloning

1. Contains an origin of replication, allowing

for replication independent of host’s
genome.
2. Contains Selective markers: Selection of
cells containing a plasmid
twin antibiotic resistance
blue-white screening
3. Contains a multiple cloning site (MCS)
4. Easy to be isolated from the host cell.
Plasmid vectors
 Bacteriophage vectors

 Advantages:
• Useful for cloning large DNA fragments
(10 - 23 kbp)
• Inherent size selection for large inserts
 Disadvantages:
• Less easy to handle
 vectors

 Left arm:
• head & tail proteins
 Right arm:
• DNA synthesis
• regulation
• host lysis
 Deleted central
region:
• integration &
excision
• regulation
Cosmid vectors

combine the properties of plasmid vectors with the useful

properties of the l cos site
 Advantages:
• Useful for cloning very large DNA fragments
(32 - 47 kbp)
• Inherent size selection for large inserts
• Handle like plasmids
 Disadvantages:
• Not easy to handle very large plasmids (~ 50 kbp)
ZAP
 BACs and YACs

BACs : Bacterial Artificial Chromosomes

YACs : Yeast Artificial Chromosomes
 Advantages:
• Useful for cloning extremely large DNA fragments
(100 - 2,000 kbp)
• This is very important for genome sequencing projects
 Disadvantages:
• Not easy to handle extremely large DNA molecules
BAC vector

 oriS and oriE mediate

replication
 parA and parB
maintain single copy
number
 ChloramphenicolR
marker

Figure 5.5
YAC vector

large
inserts

ARS URA3 HIS3

telomere centromere markers telomere
replication
origin

 Capable of carrying inserts of 200 - 2000 kbp

in yeast
 What determines the choice vector?

 insert size
 vector size
 restriction sites
 copy number
 cloning efficiency
 ability to screen for inserts
 what down-stream experiments do you plan?
Expression vector
 How to clone DNA
 Isolation of cloning vector
(bacterial plasmid) & gene-
source DNA (gene of interest)
 Insertion of gene-source DNA
into the cloning vector using
the same restriction enzyme;
bind the fragmented DNA with
DNA ligase
 Introduction of cloning vector
into cells (transformation by
bacterial cells)
 Cloning of cells (and foreign
genes)
 Identification of cell clones
carrying the gene of interest
 Screening of the clone

 The medium in this petri

dish contains the antibiotic
Kanamycin
 The bacteria on the right
contain Kanr, a plasmid
that is resistant to
Kanamycin, while the one on
the left has no resistance
 Note the difference in
growth
 Blue/White Color Screening

lacZ lacZ insert

functional enzyme nonfunctional enzyme

X-gal product X-gal product
Selecting Colonies with
Recombinant Plasmids
 Colony hybridization

 DNA probe available?

• part of same gene
• orthologue from
another species
• synthetic
oligonucleotide

Figure 6.12
End

Sayonara ………